Treatment of cachexia

ABSTRACT

The invention characterizes and provides a receptor for Proteolysis Inducing Factor (PIF) and associated methods and materials employing the same. These have utility, for example, in the provision of treatments for cachexia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/518,881 filed Dec. 14, 2009, now issued as U.S. Pat. No.8,207,310; which is a 35 USC §371 National Stage application ofInternational Application No. PCT/GB2007/0004726 filed Dec. 11, 2007;which claims the benefit under 35 USC §119(a) to Great BritainApplication Serial No. 0624687.0 filed Dec. 11, 2006. The disclosure ofeach of the prior applications is considered part of and is incorporatedby reference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the treatment of cachexia.

2. Background Information

Many patients (about 50%) with cancer suffer a severe depletion of theirlean body mass as well as adipose tissue, which has been linked with areduced survival time. Death may occur when up to 30% of body weight islost and such depletion of body mass can account to over 20% of cancerpatient deaths. This condition forms part of a complex metabolicsyndrome known as cancer cachexia, in which loss of protein fromskeletal muscle is evident, but visceral organs such as liver and kidneyare relatively unaffected, thus differentiating this condition from thatof simple starvation. Although a number of cytokines including tumornecrosis factor-a (TNF-α), interleukin-6 (IL-6) and ciliary neurotrophicfactor (CNTF) have been associated with protein depletion in cachexia,few studies have reported a direct effect on the degradative process.

The inventor has previously isolated, both from a cachexia-inducingmurine tumor and from the urine of cancer patients with cachexia, atumor factor capable of inducing weight loss in normal mice, withspecific depletion of the non-fat carcass mass (Todorov, P., Cariuk, P.,McDevitt, T., Coles, B., Fearon, K. and Tisdale, M. Characterization ofa cancer cachectic factor. Nature, 379: 739-742, 1996). The loss of bodyprotein was accounted for by an increase in protein degradation and adecrease in protein synthesis in skeletal muscle. This material, whichthey have called proteolysis inducing factor (PIF), is also capable ofinducing protein degradation in isolated gastrocnemius muscle. Theeffect of PIF on organ weights in normal mice was similar to that seenin cachexia, with a decrease in the weight of soleus and gastrocnemiusmuscles, no change in the weight of the heart or kidney and an increasein the weight of the liver.

PIF is a sulphated glycoprotein of Mr 24,000 containing both N- andO-linked oligosaccharide chains, which have been shown to be essentialfor the biological activity (Todorov, P. T., Deacon, M. and Tisdale, M.J. Structural analysis of a tumor-produced sulfated glycoprotein capableof initiating muscle protein degradation. J. Biol. Chem., 272:12279-12288, 1997). Both mouse and human PIF appear to contain identicalcarbohydrate components, as defined by their reactivity with a murinemonoclonal antibody directed towards the oligosaccharide residues. Inaddition amino acid sequence analysis of the N-terminal residues showedhomology between the two species (Cariuk, P., Lorite, M. J., Todorov, P.T., Field, W. N., Wigmore, S. J. and Tisdale, M. J. Induction ofcachexia in mice by a product isolated from the urine of cachecticcancer patients. Br. J. Cancer, 76: 606-613, 1997), suggesting thatprotein degradation in cancer cachexia may be identical in mouse andman.

In order for PIF to exert a catabolic effect on skeletal muscle theinventors believe that there must be specific cell surface receptorscapable of relaying a biological response to the intracellular proteindegradative machinery.

It is an object of the present invention to provide a high affinityreceptor for PIF; provide agents that are effective for modulatingcachexia by interacting with such receptors; and also provide a screenfor identifying such agents.

According, to a first aspect of the present invention there is providedan isolated receptor for Proteolysis Inducing Factor (PIF) characterisedin that the N terminus of the mature native receptor has the amino acidsequence:

(SEQ ID No. 1) n-DINGGGATLPQPLYQTAAVLTAGFA. or: (SEQ ID No. 13)n-DINGGGATLPQKLYLIPNVL.and functional derivatives of either.

The present invention is based upon research conducted by the inventorsinvestigating the binding activity of PIF. These experiments aredescribed in more detail in the accompanying Examples. In brief, theinventors have isolated the receptor from solubilized (1% Triton)membranes of murine myotubules by incubation with radiolabelled PIF. ThePIF-receptor complex was purified on a Wheat Germ Agglutinin-Agarosecolumn, which was capable of binding PIF, and the free receptor elutedwith 0.1M N-acetylglucosamine. The receptor was found to be a singleprotein of approximately Mr 40,000 using 15% SDS-PAGE and Sephadex G-50exclusion chromatography.

The inventors performed a tryptic digestion followed by sequenceanalysis (Edman) of the PIF receptor and established that the N-terminusof the mature receptor started with the amino acid sequence of SEQ IDNo. 1. This sequence has homology to a peptide fragment from a synovialfluid protein p205, with T-cell stimulatory activity (J. Immnuol.;(1996) 157; 1773-80). A further N-terminal sequence was obtained whichit is believed may be a polymorphic form (SEQ ID No. 13). This sequencediffers by 5 amino acids from SEQ ID No. 1 and is more basic than SEQ IDNo. 1. Although not wishing to be bound by mechanism, since PIF isacidic, it is believed that a receptor comprising SEQ ID No. 13 mayinteract more strongly with it.

Further analysis of the receptor comprising (SEQ ID No. 1 identifiedinternal peptide fragments with the following amino acid sequences:

(SEQ ID No. 2) TAINDTFLNADSNLSIGK (SEQ ID No. 3) XATVAGVSPAPANVSAAIGA(SEQ ID No. 4) . . . IIPATTAGE . . . (SEQ ID No. 5). . . TYMSPDYAAATLAG . . . (SEQ ID No. 6) FVPLPT (SEQ ID No. 7)TELSNYVTAXGTxxG (SEQ ID No. 8) VTTAGSDS (SEQ ID No. 9) DVNGG(SEQ ID No. 10) LTTWDLIADSGR.

The inventors were unable to locate any sequence homology of theseinternal peptides with other proteins in public databases. Accordinglythe PIF receptor according to the present invention represents a novelprotein.

It is preferred that the PIF receptor according to the first aspect ofthe invention further comprises at least one internal peptide sequenceof SEQ ID No. 2-10. More preferably the receptor further comprises atleast two of such internal peptides and most preferably the receptorcomprises each of the internal peptides.

Preferred functional derivatives of the PIF receptor include proteinsthat may comprise mutations (relative to the wild type) thatnevertheless do not alter the activity of the receptor. In accordancewith the present invention, preferred further changes in the receptorare commonly known as “conservative” or “safe” substitutions.Conservative amino acid substitutions are those with amino acids havingsufficiently similar chemical properties, in order to preserve thestructure and the biological function of the receptor. It is clear thatinsertions and deletions of amino acids may also be made in the abovedefined sequences without altering their function, particularly if theinsertions or deletions only involve a few amino acids, e.g., under tenand preferably under five, and do not remove or displace amino acidswhich are critical to the functional confirmation of the receptor. Theliterature provide many models on which the selection of conservativeamino acids substitutions can be performed on the basis of statisticaland physico-chemical studies on the sequence and/or the structure of anatural protein.

According to second aspect of the present invention there is provided anucleic acid encoding the receptor according to the first aspect of theinvention.

The nucleic acid may be a DNA molecule or RNA molecule (e.g., mRNA).Preferably, the nucleic acid has a nucleotide sequence substantially asset out as SEQ ID No. 11 (predicted sequence for the human N-terminalfragment SEQ ID No. 1 based on the most common codon usage) or aderivative or functional variant thereof.

(SEQ ID NO. 1) D   I   N   G   G   G   A   T   L   P (SEQ ID NO. 11)GAC ATC AAC GGC GGC GGC GCC ACC CTG CCCQ   P   L   Y   Q   T   A   A   V   LCAG CCC CTG TAC CAG ACC GCC GCC GTG CTG T   A   G   F   AACC GCC GGC TTC GCC

Alternatively, the nucleic acid has a nucleotide sequence substantiallyas set out as SEQ ID No. 14 (predicted sequence for the human N-terminalfragment SEQ ID No. 13) based on the most common codon usage) or aderivative or functional variant thereof.

(SEQ ID NO. 13) D   I   N   G   G   G   A   T   L   P (SEQ ID NO. 14)GAC ATC AAC GGC GGC GGC GCC ACC CTG CCCQ   K   L   Y   L   I   P   N   V   LCAG AAG CTG TAC CTG ATC CCC AAC GTG CTG

The nucleic acid may be contained within a suitable vector to form arecombinant vector. Hence, according to a third aspect of the inventionthere is provided a vector comprising a nucleic acid according to thesecond aspect. The vector may for example be a plasmid, cosmid or phage.Such recombinant vectors are highly useful for transforming cells withthe DNA molecule, for producing the receptor according to the firstaspect of the invention.

Recombinant vectors may also include other functional elements. Forinstance, recombinant vectors can be designed such that the vector willautonomously replicate in the cell. In this case, elements which induceDNA replication may be required in the recombinant vector.Alternatively, the recombinant vector may be designed such that thevector and nucleic acid molecule integrates into the genome of a cell.In this case DNA sequences which favour targeted integration (e.g., byhomologous recombination) are desirable. Recombinant vectors may alsohave DNA coding for genes that may be used as selectable markers in thecloning process. The recombinant vector may also further comprise apromoter or regulator to control expression of the nucleic acid asrequired.

It will be appreciated by the skilled technician that functionalderivatives of the amino acid, and nucleic acid sequences, disclosedherein, may have a sequence which has at least 30%, preferably 40%, morepreferably 50%, and even more preferably, 60% sequence identity with theamino acid/polypeptide/nucleic acid sequences of any of the sequencesreferred to herein. An amino acid/polypeptide/nucleic acid sequence witha greater identity than preferably 65%, more preferably 75%, even morepreferably 85%, and even more preferably 90% to any of the sequencesreferred to is also envisaged. Preferably, the aminoacid/polypeptide/nucleic acid sequence has 92% identity, even morepreferably 95% identity, even more preferably 97% identity, even morepreferably 98% identity and, most preferably, 99% identity with any ofthe referred to sequences.

Calculation of percentage identities between different aminoacid/polypeptide/nucleic acid sequences may be carried out as follows. Amultiple alignment is first generated by the ClustalX program (pair wiseparameters: gap opening 10.0, gap extension 0.1, protein matrix Gonnet250, DNA matrix IUB; multiple parameters: gap opening 10.0, gapextension 0.2, delay divergent sequences 30%, DNA transition weight 0.5,negative matrix off, protein matrix gonnet series, DNA weight IUB;Protein gap parameters, residue-specific penalties on, hydrophilicpenalties on, hydrophilic residues GPSNDQERK, gap separation distance 4,end gap separation off). The percentage identity is then calculated fromthe multiple alignment as (N/T)*100, where N is the number of positionsat which the two sequences share an identical residue, and T is thetotal number of positions compared. Alternatively, percentage identitycan be calculated as (N/S)*100 where S is the length of the shortersequence being compared. The amino acid/polypeptide/nucleic acidsequences may be synthesised de novo, or may be native aminoacid/polypeptide/nucleic acid sequence, or a derivative thereof.

Alternatively, a substantially similar nucleotide sequence will beencoded by a sequence which hybridizes to any of the nucleic acidsequences referred to herein or their complements under stringentconditions. By stringent conditions, we mean the nucleotide hybridisesto filter-bound DNA or RNA in 6× sodium chloride/sodium citrate (SSC) atapproximately 45° C. followed by at least one wash in 0.2×SSC/0.1% SDSat approximately 5-65° C. Alternatively, a substantially similarpolypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100amino acids from the peptide sequences according to the presentinvention.

Due to the degeneracy of the genetic code, it is clear that any nucleicacid sequence could be varied or changed without substantially affectingthe sequence of the receptor protein encoded thereby, to provide afunctional variant thereof. Suitable nucleotide variants are thosehaving a sequence altered by the substitution of different codons thatencode the same amino acid within the sequence, thus producing a silentchange. Other suitable variants are those having homologous nucleotidesequences but comprising all, or portions of, sequence which are alteredby the substitution of different codons that encode an amino acid with aside chain of similar biophysical properties to the amino acid itsubstitutes, to produce a conservative change. For example smallnon-polar, hydrophobic amino acids include glycine, alanine, leucine,isoleucine, valine, proline, and methionine. Large non-polar,hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.The polar neutral amino acids include serine, threonine, cysteine,asparagine and glutamine. The positively charged (basic) amino acidsinclude lysine, arginine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

The accurate alignment of protein or DNA sequences is a complex process,which has been investigated in detail by a number of researchers. Ofparticular importance is the trade-off between optimal matching ofsequences and the introduction of gaps to obtain such a match. In thecase of proteins, the means by which matches are scored is also ofsignificance. The family of PAM matrices (e.g., Dayhoff, M. et al.,1978, Atlas of protein sequence and structure, Natl. Biomed. Res.Found.) and BLOSUM matrices quantify the nature and likelihood ofconservative substitutions and are used in multiple alignmentalgorithms, although other, equally applicable matrices will be known tothose skilled in the art. The popular multiple alignment programClustalW, and its windows version ClustalX (Thompson et al., 1994,Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, NucleicAcids Research, 24, 4876-4882) are efficient ways to generate multiplealignments of proteins and DNA.

Frequently, automatically generated alignments require manual alignment,exploiting the trained user's knowledge of the protein family beingstudied, e.g., biological knowledge of key conserved sites. One suchalignment editor programs is Align(http://www.gwdg.de/˜dhepper/download/; Hepperle, D., 2001: MulticolorSequence Alignment Editor. Institute of Freshwater Ecology and InlandFisheries, 16775 Stechlin, Germany), although others, such as JalView orCinema are also suitable.

Calculation of percentage identities between proteins occurs during thegeneration of multiple alignments by Clustal. However, these values needto be recalculated if the alignment has been manually improved, or forthe deliberate comparison of two sequences. Programs that calculate thisvalue for pairs of protein sequences within an alignment includePROTDIST within the PHYLIP phylogeny package (Felsenstein;http://evolution.gs.washington.edu/phylip.html) using the “SimilarityTable” option as the model for amino acid substitution (P). For DNA/RNA,an identical option exists within the DNADIST program of PHYLIP.

Other modifications in protein sequences are also envisaged and withinthe scope of the claimed invention, i.e., those which occur during orafter translation, e.g., by acetylation, amidation, carboxylation,phosphorylation, proteolytic cleavage or linkage to a ligand.

It will be appreciated that knowledge of the receptor according to thefirst aspect of the invention enabled the inventors to develop agentsthat modulate the activity of the receptor and thereby clinically managecachexia. The present invention also provides a medical use for suchagents. Thus, according to a fourth aspect of the invention, there isprovided an agent that decreases the biological activity of the PIFreceptor according to the first aspect of the invention for use as amedicament.

According to a fifth aspect of the invention there is provided the useof an agent that decreases the biological activity of the PIF receptorin the manufacture of a medicament for the treatment of cachexia.

According to a sixth aspect of the present invention, there is provideda method for the treatment of cachexia comprising administering to asubject in need of such treatment a therapeutically effective amount ofan agent that decreases the biological activity of the PIF receptor.

Agents capable of decreasing the biological activity of PIF may achievetheir effect by a number of means. For instance, such agents may:

-   -   (a) decrease the expression of the PIF receptor;    -   (b) increase receptor desensitisation or receptor breakdown;    -   (c) reduce interaction between PIF and its endogenous receptor;    -   (d) reduces PIF receptor mediated intracellular signalling;    -   (e) competes with endogenous PIF receptors for PIF binding;    -   (f) binds to the PIF receptor to block PIF binding; or    -   (g) binds to PIF preventing interaction with the receptor.

It is preferred that the agent directly interacts with the receptor ofPIF (i.e., (e) and (f) above) or it acts to inhibit transcription ortranslation of the PIF receptor (a) above. It will be appreciated thatsuch agents may only be designed in the light of knowledge of thesequence of the PIF receptor according to the first aspect of theinvention.

A preferred agent for use according to the fourth, fifth or sixthaspects of the present invention is a neutralising antibody raisedagainst the PIF receptor. Such antibodies represent an important featureof the invention. Thus, according to a seventh aspect of the invention,there is provided an antibody, or a functional derivative thereof,against the PIF receptor according to the first aspect of the invention.

The antibody preferably blocks PIF receptor mediated intracellularsignalling. This may be by blocking the ligand binding site on thereceptor or may dissociate the receptor from its signal transductionpathway by some allosteric means.

Antibodies according to the invention may be produced as polyclonal seraby injecting antigen into animals. Preferred polyclonal antibodies maybe raised by inoculating an animal (e.g., a rabbit) with antigen usingtechniques known to the art. The antigen may be the whole PIF receptor(in glycosylated or non-glycosylated form) or a fragment thereof.Preferred fragments for generating the antibodies are peptides with SEQID No. 1-10 or 13. A preferred polyclonal antibody is raised against thepeptide of SEQ ID No.1 as described in the Examples. Another preferredpolyclonal antibody is raised against the peptide of SEQ ID No. 13.

Alternatively the antibody may be monoclonal and raised in mice.Conventional hybridoma techniques may be used to raise the antibodies.The antigen used to generate monoclonal antibodies according to thepresent invention may be the whole PIF receptor (in glycosylated ornon-glycosylated form) or a fragment thereof. Preferred fragments forgenerating the antibodies are peptides with SEQ ID No. 1-10 or 13.

It is preferred that the antibody is a γ-immunoglobulin (IgG).

It will be appreciated that the variable region of an antibody definesthe specificity of the antibody and as such this region should beconserved in functional derivatives of the antibody according to theinvention. The regions beyond the variable domains (C-domains) arerelatively constant in sequence. It will be appreciated that thecharacterising feature of antibodies according to the invention is theV_(H) and V_(L) domains. It will be further appreciated that the precisenature of the C_(H) and C_(L) domains is not, on the whole, critical tothe invention. In fact preferred antibodies according to the inventionmay have very different C_(H) and C_(L) domains. Furthermore, asdiscussed more fully below, preferred antibody functional derivativesmay comprise the Variable domains without a C-domain (e.g., scFVantibodies).

The inventors have found that antibodies, or functional derivativesthereof, according to the seventh aspect of the invention havesurprising efficacy for preventing the development of cachexia in cancerpatients.

An antibody derivative may have 75% sequence identity, more preferably90% sequence identity and most preferably has at least 95% sequenceidentity to a monoclonal antibody or specific antibody in a polyclonalmix. It will be appreciated that most sequence variation may occur inthe framework regions (FRs) whereas the sequence of the CDRs of theantibodies, and functional derivatives thereof, is most conserved.

A number of preferred embodiments of the seventh aspect of the inventionrelate to molecules with both Variable and Constant domains. However itwill be appreciated that antibody fragments (e.g., scFV antibodies) arealso encompassed by the invention that comprise essentially the Variableregion of an antibody without any Constant region.

Antibodies generated in one species are known to have several seriousdrawbacks when used to treat a different species. For instance whenmurine antibodies are used in humans they tend to have a shortcirculating half-life in serum and are recognised as foreign proteins bythe patient being treated. This leads to the development of an unwantedhuman anti-mouse (or rat) antibody response. This is particularlytroublesome when frequent administrations of the antibody is required asit can enhance the clearance thereof, block its therapeutic effect, andinduce hypersensitivity reactions. Accordingly preferred antibodies (ifof non-human source) for use in human therapy are humanised.

Monoclonal antibodies are generated by the hybridoma technique whichusually involves the generation of non-human mAbs. The technique enablesrodent monoclonal antibodies with almost any specificity to be produced.Accordingly preferred embodiments of the invention may use such atechnique to develop monoclonal antibodies against the PIF receptor.Although such antibodies are useful therapeutically, it will beappreciated that such antibodies are not ideal therapeutic agents inhumans (as suggested above). Ideally, human monoclonal antibodies wouldbe the preferred choice for therapeutic applications. However, thegeneration of human mAbs using conventional cell fusion techniques hasnot to date been very successful. The problem of humanisation may be atleast partly addressed by engineering antibodies that use V regionsequences from non-human (e.g., rodent) mAbs and C region (and ideallyFRs from V region) sequences from human antibodies. The resulting‘engineered’ mAbs are less immunogenic in humans than the rodent mAbsfrom which they were derived and so are better suited for clinical use.

Humanised antibodies may be chimaeric monoclonal antibodies, in which,using recombinant DNA technology, rodent immunoglobulin constant regionsare replaced by the constant regions of human antibodies. The chimaericH chain and L chain genes may then be cloned into expression vectorscontaining suitable regulatory elements and induced into mammalian cellsin order to produce fully glycosylated antibodies. By choosing anappropriate human H chain C region gene for this process, the biologicalactivity of the antibody may be pre-determined. Such chimaericantibodies are superior to non-human monoclonal antibodies in that theirability to activate effector functions can be tailored for a specifictherapeutic application, and the anti-globulin response they induce isreduced.

Such chimaeric molecules are preferred agents for treating cachexiaaccording to the present invention. RT-PCR may be used to isolate theV_(H) and V_(L) genes from preferred mAbs, cloned and used to constructa chimaeric version of the mAb possessing human domains.

Further humanisation of antibodies may involve CDR-grafting or reshapingof antibodies. Such antibodies are produced by transplanting the heavyand light chain CDRs of a rodent mAb (which form the antibody's antigenbinding site) into the corresponding framework regions of a humanantibody.

A further preferred agent for use according to the fourth, fifth orsixth aspects of the present invention is a soluble PIF receptor or afunctional derivative or fragment thereof according to the first aspectof the invention. The PIF receptor is an integral protein of the plasmamembrane. The inventors have found that soluble receptors according tothe first aspect of the invention may be introduced into a target tissueand will compete for endogenous PIF. PIF binding to the soluble receptorwill not exert a physiological effect because the soluble receptor isnot linked to an intracellular signalling pathway. Accordingly suchagents are effective for reducing PIF receptor mediated cachexia.Peptide fragments of the PIF receptor may also be used as agentsaccording to the invention. The inventors believe that the PIF bindingsite on the receptor may be in the terminal portion. It is thereforepreferred that the agent is an N terminal fragment of the PIF receptor.For instance the agent may be the peptide of SEQ ID NO. 1 (see Example3) or SEQ ID NO. 13.

Derivatives of peptide agents used according to the invention includederivatives that increase the half-life of the agent in vivo. Examplesof derivatives capable of increasing the half-life of polypeptidesaccording to the invention include peptoid derivatives, D-amino acidderivatives and peptide-peptoid hybrids.

Proteins and peptide agents according to the present invention may besubject to degradation by a number of means (such as protease activityat a target site). Such degradation may limit their bioavailability andhence therapeutic utility. There are a number of well-establishedtechniques by which peptide derivatives that have enhanced stability inbiological contexts can be designed and produced. Such peptidederivatives may have improved bioavailability as a result of increasedresistance to protease-mediated degradation. Preferably, a derivativesuitable for use according to the invention is more protease-resistantthan the protein or peptide from which it is derived.Protease-resistance of a peptide derivative and the protein or peptidefrom which it is derived may be evaluated by means of well-known proteindegradation assays. The relative values of protease resistance for thepeptide derivative and peptide may then be compared.

Peptoid derivatives of proteins and peptides according to the inventionmay be readily designed from knowledge of the structure of the receptoraccording to the first aspect of the invention or an agent according tothe fourth, fifth or sixth aspect of the invention. Commerciallyavailable software may be used to develop peptoid derivatives accordingto well-established protocols.

Retropeptoids, (in which all amino acids are replaced by peptoidresidues in reversed order) are also able to mimic proteins or peptidesaccording to the invention. A retropeptoid is expected to bind in theopposite direction in the ligand-binding groove, as compared to apeptide or peptoid-peptide hybrid containing one peptoid residue. As aresult, the side chains of the peptoid residues are able to point in thesame direction as the side chains in the original peptide.

A further embodiment of a modified form of peptides or proteinsaccording to the invention comprises D-amino acid forms. In this case,the order of the amino acid residues is reversed. The preparation ofpeptides using D-amino acids rather than L-amino acids greatly decreasesany unwanted breakdown of such derivative by normal metabolic processes,decreasing the amounts of the derivative which needs to be administered,along with the frequency of its administration.

A further preferred agent according to the fourth, fifth or sixthaspects of the invention is an antisense DNA or RNA molecule that willbind to endogenous PIF receptor transcripts. Such antisense moleculesreduce PIF receptor expression and thereby reduce PIF mediated activity.Preferred antisense molecules represent the antisense of nucleic acidsaccording to the second aspect of the invention. By way of example, thesequence of an antisense molecule against the receptor would be:

(SEQ ID NO. 12) 5′GGCGAAGCCGGCGGTCAGCACGGCGGCGGTCTGGTACAGGGGCTGGGGCAGGGTGGCGCCGCCGCC GTTGATGTC . . . 3′.

The reverse-complement molecule of SEQ ID NO. 12 acts as antisense tothe N-terminal 25 amino acids of SEQ ID NO. 1.

(SEQ ID NO. 15) 5′CAGCACGTTGGGGATCAGGTACAGCTTCTGGGGCAGGGTGGCGCCGCCGCCGTTGATGTC . . . 3′.

The reverse-complement molecule of SEQ ID NO. 15 acts as antisense tothe N-terminal 20 amino acids of SEQ ID NO. 13.

siRNA may also be used as an agent according to the invention. siRNAforms part of a gene silencing mechanism, known as RNA interference(RNAi) which results in the sequence-specific destruction of mRNAs andenables a targeted knockout of gene expression. siRNA used in genesilencing may comprise double stranded RNA of 21 nucleotides length,typically with a 2-nucleotide overhang at each 3′ end. Alternatively,short hairpin RNAs (shRNAs) using sense and antisense sequencesconnected by a hairpin loop may be used. Both siRNAs and shRNAs can beeither chemically synthesized and introduced into cells for transientRNAi or expressed endogenously from a promoter for long-term inhibitionof gene expression. siRNA molecules for use as an agent according to theinvention may comprise either double stranded RNA of 10-50 nucleotides.Preferably, siRNAs for use as an agent according to the inventioncomprise 18-30 nucleotides. More preferably, siRNAs for use as an agentaccording to the invention comprise 21-25 nucleotides. And mostpreferably, siRNAs for use as an agent according to the inventioncomprise 21 nucleotides. It will be appreciated that siRNAs will need tobe based upon the sequences according to the second aspect of theinvention. Preferred double stranded siRNA molecules comprise a sensestrand of 21-25 contiguous nucleotides from SEQ ID NO. 11 bound to thecomplementary antisense strand (e.g., as defined in SEQ. ID NO. 12).Alternatively, shRNAs using sense and antisense sequences may be used asan agent according to the invention. Preferably, shRNAs using sense andantisense sequences that may be employed as an agent according to theinvention comprise 20-100 nucleotides. More preferably, shRNAs usingsense and antisense sequences that may be employed as an agent accordingto the invention comprise 42 nucleotides and may comprise 21 nucleotidesfrom SEQ ID NO. 11 linked to the complementary 21 nucleotides from SEQID NO. 12 (or SEQ ID NO. 14 linked to 15). As for siRNAs, it will beappreciated that shRNAs will need to be based upon the sequencesaccording to the second aspect of the invention.

The inventor has realised that PIF binds to its receptor througholigosaccharide chains. Accordingly, other preferred agents have asimilar configuration of oligosaccharides to those found on PIF and/orits receptor (e.g., chondroitin sulphate or other sulphoidoligosaccharides). Such agents compete with PIF for binding to thereceptor and are therefore useful for reducing cachexia.

Agents according to the fourth, fifth or sixth aspects of the inventionmay take a number of different forms depending, in particular on themanner in which the composition is to be used. Thus, for example, acomposition comprising the agent may be in the form of a powder, tablet,capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray,micelle, transdermal patch, liposome or any other suitable form that maybe administered to a person or animal. It will be appreciated that thevehicle of the composition of the invention should be one which is welltolerated by the subject to whom it is given and enables delivery of thecompounds to the brain.

The composition of the invention may be used in a number of ways. Forinstance, systemic administration may be required in which case theagent may be contained within a composition which may, for example, beingested orally in the form of a tablet, capsule or liquid.Alternatively the composition may be administered by injection into theblood stream. Injections may be intravenous (bolus or infusion) orsubcutaneous (bolus or infusion) or intramuscularly. The agents may beadministered by inhalation (e.g., intranasally). The agents may also beadministered centrally, by means of intracerebral,intracerebroventricular or intrathecal delivery.

The agents may also be incorporated within a slow or delayed releasedevice. Such devices may, for example, be inserted on or under the skinand the agent may be released over weeks or even months. Such a devicemay be particularly useful for chronically ill patients. The devices maybe particularly advantageous when an agent is used which would normallyrequire frequent administration (e.g., at least daily ingestion of atablet or daily injection).

It will be appreciated that the amount of an agent required isdetermined by biological activity and bioavailability which in turndepends on the mode of administration, the physicochemical properties ofthe agent employed and whether it is being used as a monotherapy or in acombined therapy. The frequency of administration will also beinfluenced by the abovementioned factors and particularly the half-lifeof the agent within the subject being treated.

Optimal dosages to be administered may be determined by those skilled inthe art, and will vary with the particular agent in use, the strength ofthe preparation, the mode of administration, and the advancement of thedisease condition (e.g., the severity of the cachexia or event the stageof cancer development). Additional factors depending on the particularsubject being treated will result in a need to adjust dosages, includingsubject age, weight, gender, diet, and time of administration.

Known procedures, such as those conventionally employed by thepharmaceutical industry (e.g., in vivo experimentation, clinical trials,etc.), may be used to establish specific formulations of compositionsand precise therapeutic regimes (such as daily doses of the compoundsand the frequency of administration).

Generally, a daily dose of between 0.01 μg/kg of body weight and 1.0g/kg of body weight of an agent (e.g., a soluble receptor based on SEQID NO. 1) may be used for the treatment of cachexia. The amount usedwill depend upon which specific agent is used. More preferably, thedaily dose is between 0.01 mg/kg of body weight and 100 mg/kg of bodyweight.

Daily doses may be given as a single administration (e.g., a dailytablet for oral consumption or as a single daily injection).Alternatively, the agent used may require administration twice or moretimes during a day. As an example, an agent may be administered as two(or more depending upon the severity of the cachexia) daily doses ofbetween 25 mgs and 5000 mgs in tablet form. A patient receivingtreatment may take a first dose upon waking and then a second dose inthe evening (if on a two dose regime) or at 3 or 4 hourly intervalsthereafter. Alternatively, a slow release device may be used to provideoptimal doses to a patient without the need to administer repeateddoses.

The use of an antibody raised against the PIF receptor as an agentaccording to the invention may involve the administration thereof as aweekly, twice weekly or thrice weekly dose (or more depending upon theseverity of the cachexia) of between 25 mgs and 5000 mgs in injectableform. Alternatively, a slow release device may be used to provideoptimal doses to a patient without the need to administer repeateddoses.

This invention further provides a pharmaceutical composition comprisinga therapeutically effective amount of an agent of the invention and apharmaceutically acceptable vehicle. In one embodiment, the amount ofthe agent (e.g, a soluble receptor) is an amount from about 0.01 mg toabout 800 mg. In another embodiment, the amount is from about 0.01 mg toabout 500 mg.

In a further embodiment, the vehicle is a liquid and the composition isa solution. In another embodiment, the vehicle is a solid and thecomposition is a tablet. In a further embodiment, the vehicle is a geland the composition is a suppository.

Agents are preferably combined with a pharmaceutically acceptablevehicle prior to administration.

In the subject invention a “therapeutically effective amount” is anyamount of an agent which, when administered to a subject suffering froma disease against which the agent is effective, causes reduction,remission, or regression of the cachexia through the preservation oflean body mass. A “subject” is a vertebrate, mammal, domestic animal orpreferably a human being.

In the practice of this invention a “pharmaceutically acceptablevehicle” is any physiological vehicle known to those of ordinary skillin the art useful in formulating pharmaceutical compositions.

In one embodiment, the pharmaceutical vehicle may be a liquid and thepharmaceutical composition would be in the form of a solution. Inanother embodiment, the pharmaceutically acceptable vehicle is a solidand the composition is in the form of a powder or tablet. In a furtherembodiment, the pharmaceutical vehicle is a gel and the composition isin the form of a suppository or cream. In a further embodiment the agentor composition may be formulated as a part of a pharmaceuticallyacceptable transdermal patch.

A solid vehicle can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the vehicle is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with avehicle having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid vehicles include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid vehicles are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidvehicle such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid vehicle can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid vehicles fororal and parenteral administration include water (partially containingadditives as above, e.g., cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g., glycols) and their derivatives,and oils (e.g., fractionated coconut oil and arachis oil). Forparenteral administration, the vehicle can also be an oily ester such asethyl oleate and isopropyl myristate. Sterile liquid vehicles are usefulin sterile liquid form compositions for parenteral administration. Theliquid vehicle for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by for example, intramuscular, intrathecal,epidural, intraperitoneal or subcutaneous injection.

Sterile solutions can also be administered intravenously. The agents maybe prepared as a sterile solid composition which may be dissolved orsuspended at the time of administration using sterile water, saline, orother appropriate sterile injectable medium. Vehicles are intended toinclude necessary and inert binders, suspending agents, lubricants,flavorants, sweeteners, preservatives, dyes, and coatings.

The agents can be administered orally in the form of a sterile solutionor suspension containing other solutes or suspending agents (forexample, enough saline or glucose to make the solution isotonic), bilesalts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleateesters of sorbitol and its anhydrides copolymerized with ethylene oxide)and the like.

An agent can also be administered orally either in liquid or solidcomposition form. Compositions suitable for oral administration includesolid forms, such as pills, capsules, granules, tablets, and powders,and liquid forms, such as solutions, syrups, elixirs, and suspensions.Forms useful for parenteral administration include sterile solutions,emulsions, and suspensions.

The agents may be combined with a pharmaceutically acceptable vehicleand another therapeutically active agent prior to administration. Theother therapeutically active agent may be for the treatment of cancer orcachexia.

Knowledge of the PIF receptor has enabled the inventors to develop ascreen for identifying whether or not test compounds are putative agentsfor use according to the fourth, fifth or sixth aspects of theinvention. Thus, according to a eighth aspect of the present inventionthere is provided a method of screening a compound to test whether ornot the compound has efficacy for treating cachexia, comprising:

-   -   (i) exposing cells or membranes comprising PIF receptors        according to the first aspect of the invention to a test        compound for a predetermined length of time;    -   (ii) detecting the activity or expression of the PIF receptor;        and    -   (iii) comparing the activity or expression of the PIF receptors        in the cells or membranes treated with the compound relative to        activity or expression found in control cells or membranes that        were not treated with the compound

wherein compounds with efficacy for treating cachexia decrease activityor decrease expression of the PIF receptor relative to the controls.

It will be appreciated that the method according to the eighth aspect ofthe invention may be adapted such that it is used to test whether or nota compound causes cachexia. Therefore according to a ninth aspect of theinvention there is provided a method of screening a compound, to testwhether or not the compound causes cachexia, comprising:

-   -   (i) exposing cells or membranes comprising PIF receptors        according to the first aspect of the invention to a test        compound for a predetermined length of time;    -   (ii) detecting the activity or expression of the PIF receptor;        and    -   (iii) comparing the activity or expression of the PIF receptors        in the cells or membranes treated with the compound relative to        activity or expression found in control cells or membranes that        were not treated with the compound

wherein compounds that cause cachexia increase activity or increaseexpression of the PIF receptor relative to the controls.

With regards to “detecting the activity or expression of the PIFreceptor” according to the eighth and the ninth aspects of the presentinvention, by “activity” of the PIF receptor we mean the detection ofligand-receptor binding; detection of receptor-mediated intracellularsignal transduction; or determination of an end-point physiologicaleffect. By “expression” we mean detection of the receptor protein eitherin the cell membrane, the Endoplasmatic Reticulum or the GolgiApparatus; or detection of the mRNA encoding the receptor protein.

The screening methods of the invention are based upon the inventors'realisation that the extent of PIF receptor expression and/or activitymay be closely related to the development of cachexia.

Cells used according to the eighth or ninth aspects of the invention maybe contained within an experimental animal (e.g., a mouse or rat) whenthe method is an in vivo based test. Alternatively the cells may be in atissue sample (for ex vivo based tests) or the cells may be grown inculture. It will be appreciated that such cells should express, or maybe induced to express, functional PIF receptor.

It is also possible to use cells that are not naturally predisposed toexpress PIF receptor provided that such cells are transformed with anexpression vector according to the third aspect of the invention. Suchcells represent preferred test cells for use according to the invention.This is because animal cells or even prokaryotic cells may betransformed to express human PIF receptor and therefore represent a goodcell model for testing the efficacy of candidate drugs for use in humantherapy.

The methods according to the eighth and ninth aspects of the inventionmay also be based upon the use of cell membranes comprising the PIFreceptor or the isolated soluble PIF receptor. Such membranes arepreferably derived from the above described cells. Such membranes maynot comprise functional PIF receptors but may be prepared such that themembranes may be used in receptor binding based methods.

The activity or expression of PIF receptors may be measured using anumber of conventional techniques.

The test may be an immunoassay based test. For instance, labelledantibodies (preferably a labelled antibody according to the seventhaspect of the invention) may be used in an immunoassay to evaluatereceptor levels in cells or cell membranes. Such tests are particularlyuseful when evaluating whether or not an agents modulates PIF receptorexpression, degradation or desentisation (i.e., receptor recycling).Antibodies raised against the ligand binding site may also be used toevaluate whether or not the test compound is an agonist or antagonist ofthe PIF receptor.

Alternatively conventional receptor binding assays (e.g., usingradiolabelled PIF ligand and/or radiolabelled test compounds) may beemployed. Such an assay may involve exposing membranes comprising thePIF receptor to various concentrations of [³⁵S]PIF in the absence orpresence of a competing test compound. Bound and free radioactivity maybe counted by separation of membranes from buffer by centrifugation ormembrane harvesting on filters. A preferred receptor binding based assayis described in the Examples.

Alternatively a functional activity measuring PIF activity may beemployed. For instance the development of cachexia may be monitored in atest animal.

Furthermore molecular biology techniques may be used to detect the PIFreceptor. For instance, cDNA may be generated from mRNA extracted fromtested cells or subject and primers designed to amplify test sequencesused in a quantitative Polymerase Chain Reaction to amplify from cDNA.

When a subject is used (e.g., an animal model or even an animal modelengineered to express human PIF receptor), the test compound should beadministered to the subject for a predetermined length of time and thena sample taken from the subject for assaying PIF receptor activity orexpression. The sample may for instance be blood or biopsy tissue.However the assay may be functional in which case the end point may bethe monitoring of cachexia in the test subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be illustrated further by Example and with referenceto the following drawings, in which:

FIG. 1 shows binding of [³H]peptide PIF to C₂C₁₂ membranes withouttreatment () or after incubation with PN Gase F (▪) or 0-glycosidase(▴) for 24 h;

FIG. 2A shows the results of electrophoresis of C₂C₁₂ membranes whichwere solubilized in 1% Triton and 50 μg of protein was loaded into eachwell of a 15% SDS polyacrylamide gel. Samples were electrophoresed andtransferred electrophoretically to a nitrocellulose filter which hadbeen preblocked overnight in blocking buffer (5% Marvel in PBScontaining 0.1% Tween-20). The filters were blotted with increasingconcentrations of [³⁵S]PIF in blocking bluffer for 2 h at roomtemperature. The concentrations of [³⁵S]PIF were lane 1, 5 nM; lane 2,10 nM; lane 3, 15 nM; lane 4, 30 nM; lane 5, 60 nM; lane 6, 80 nM andlane 7, 100 nM. The filter was washed three times with PBS containing0.1% Tween-20, air dried and processed for autoradiography. The 40 kDabands were excised from each lane and counted directly for radioactivity(19). FIG. 2B shows a recording of a nitrocellulose filter onto whichmembrane proteins which were transferred electrophoretically and blottedwith [³⁵S]PIF (30 nM), alone, lane 1 or together with 10 nM, lane 2; 20nM, lane 3; 40 nM, lane 4; 80 nM, lane 5; 160 nM, lane 6 and 320 nM,lane 7 unlabeled PIF in blocking buffer for 2 h at room temperature andprocessed for autoradiography;

FIG. 3 shows isolated radioactive fractions of C₂C₁₂ membrane which wereelectrophoresed on 15% SDS-PAGE where the receptor appeared as a singleprotein of apparent Mr 40 kDa (1—Molecular weight markers,2,3,4—Fractions from column);

FIG. 4 shows a graphical representation of protein degradation in C₂C₁₂myotubes in response to PIF in the absence (▪) and presence (

) of the N-terminal fragment of the PIF receptor (10 μM); Statisticalanalysis: Difference in means between groups was determined by one-wayANOVA, followed by Tukey's post-test.

Difference from control is indicated as a, p<0.05; b, p<0.01 and c,p<0.001. Differences between {circle around (R )} peptide group andcontrol group are indicated as d, p<0.05; e, p<0.01 and f, p<0.001;

FIG. 5 shows a graphical representation of chymotrypsin-like enzymeactivity in C₂C₁₂ myotubes in response to PIF in the absence (□) andpresence (▪) of the N-terminal fragment of the PIF receptor (10 μM);Statistical analysis: Difference in means between groups was determinedby one-way ANOVA, followed by Tukey's post-test. Difference from controlis indicated as a, p<0.05 and difference between {circle around (R )}peptide group and control is indicated as d, p<0.05;

FIG. 6A shows a graphical representation of expression of 20S proteasomeα-subunits in C₂C₁₂ myotubes in response to PIF and the PIF receptorpeptide (Lane legend: 1 PBS control, 2 PIF 2.1 nM, 3 PIF 4.2 nM, 4 PIF10.5 nM, 5 PIF 16.8 nM, 6 {circle around (R )} control, 7 {circle around(R )} and PIF 2.1 nM, 8 {circle around (R )} and PIF 4.2 nM, 9 {circlearound (R )} and PIF 10.5 nM, 10 {circle around (R )} and PIF 16.8 nM);Statistical analysis: Difference in means between groups was determinedby one-way ANOVA, followed by Tukey's post-test. Difference from controlis indicated as a, p<0.05. Differences between {circle around (R )}peptide group and control group are indicated as d, p<0.05 and e,p<0.01;

FIG. 6B shows a graphical representation of expression of MSS1 in C₂C₁₂myotubes in response to PIF and the PIF receptor peptide (Lane legend: 1PBS control, 2 PIF 2.1 nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM, 5 PIF 16.8 nM, 6{circle around (R )} control, 7 {circle around (R )} and PIF 2.1 nM, 8{circle around (R )} and PIF 4.2 nM, 9 {circle around (R )} and PIF 10.5nM, 10 {circle around (R )} and PIF 16.8 nM); Statistical analysis:Difference in means between groups was determined by one-way ANOVA,followed by Tukey's post-test. Difference from control is indicated asb, p<0.05. Differences between {circle around (R )} peptide group andcontrol group are indicated as e, p<0.01 and f, p<0.001;

FIG. 6C shows a graphical representation of expression of p42 in C₂C₁₂myotubes in response to PIF and the PIF receptor peptide (Lane legend: 1PBS control, 2 PIF 2.1 nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM, 5 PIF 16.8 nM, 6{circle around (R )} control, 7 {circle around (R )} and PIF 2.1 nM, 8{circle around (R )} and PIF 4.2 nM, 9 {circle around (R )} and PIF 10.5nM, 10 {circle around (R )} and PIF 16.8 nM); Statistical analysis:Difference in means between groups was determined by one-way ANOVA,followed by Tukey's post-test. Difference from control is indicated asa, p<0.05. Differences between {circle around (R )} peptide group andcontrol group are indicated as d, p<0.05 and e, p<0.01;

FIG. 6D shows a graphical representation of expression of E2_(14k) inC₂C₁₂ myotubes in response to PIF and the PIF receptor peptide (Lanelegend: 1 PBS control, 2 PIF 2.1 nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM, 5 PIF16.8 nM, 6 {circle around (R )} control, 7 {circle around (R )} and PIF2.1 nM, 8 {circle around (R )} and PIF 4.2 nM, 9 {circle around (R )}and PIF 10.5 nM, 10 {circle around (R )} and PIF 16.8 nM); Statisticalanalysis Difference in means between groups was determined by one-wayANOVA, followed by Tukey's post-test. Difference from control isindicated as b, p<0.01. Differences between peptide group and controlgroup are indicated as e, p<0.01;

FIG. 6E shows a representation of Myosin expression in C₂C₁₂ myotubes inresponse to PIF and the PIF receptor peptide (Lane legend: 1 PBScontrol, 2 PIF 2.1 nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM, 5 PIF 16.8 nM, 6{circle around (R )} control, 7 {circle around (R )} and PIF 2.1 nM, 8{circle around (R )} and PIF 4.2 nM, 9 {circle around (R )} and PIF 10.5nM, 10 {circle around (R )} and PIF 16.8 nM); Statistical analysis:Difference in means between groups was determined by one-way ANOVA,followed by Tukey's post-test. Difference from control is indicated asa, p<0.05. Differences between peptide group and control group areindicated as d, p<0.05 and e, p<0.01.

FIG. 7 shows a photograph of a Western Blot using anti-receptor antiseraperformed after purification of the antibody by adding 50% saturatedammonium sulphate followed by Protein-A column chromatography:

-   -   1 Purified receptor 5 μg protein    -   2 Purified receptor 10 μg protein    -   3 Purified receptor 20 μg protein    -   4 Crude membrane fraction 20 μg protein    -   5 Crude membrane fraction 30μg protein    -   6 Crude membrane fraction 40 μg protein

FIG. 8 shows a graphic representation of the effect of the antibody tothe PIF receptor at concentrations between 5 and 15 μg/ml on proteindegradation in vitro induced by PIF; Statistical analysis: Difference inmeans between groups was determined by one-way ANOVA, followed byTukey's post-test. Difference from control is indicated as b, p<0.01,differences between control and anti-receptor are indicated as d, p<0.05and e, p<0.01;

FIG. 9 shows graphic representation of the effect of the antibody to thePIF receptor on the PIF-induced increase in chymotrypsin-like enzymeactivity; Statistical analysis: Difference in means between groups wasdetermined by one-way ANOVA, followed by Tukey's post-test. Differencesfrom control is indicated as c, p<0.001, differences between control andanti-receptor are indicated as d, p<0.05 and e, p<0.01;

FIG. 10A shows a graphical representation of expression of 20Sproteasome α-subunits in C₂C₁₂ myotubes in response to PIF andanti-receptor antibody (10 μg/ml) (Lane legend: 1 PBS control, 2 PIF 2.1nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM, 5 PIF 16.8 nM, 6 αPIF Ab control, 7αPIF Ab and PIF 2.1 nM, 8 αPIF AB and PIF 4.2 nM, 9 αPIF Ab and PIF 10.5nM, 10 αPIF Ab {circle around (R )} and PIF 16.8 nM); Statisticalanalysis: Difference in means between groups was determined by one-wayANOVA, followed by Tukey's post-test. Differences from control areindicated as c, p<0.001. Differences between anti-receptor 10 mg/ml andPIF are indicated as f, p<0.001;

FIG. 10B shows a graphical representation of expression of MSS1 in C₂C₁₂myotubes in response to PIF and anti-receptor antibody (10 μg/ml) (Lanelegend: 1 PBS control, 2 PIF 2.1 nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM, 5 PIF16.8 nM, 6 αPIF Ab control, 7 αPIF Ab and PIF 2.1 nM, 8 αPIF AB and PIF4.2 nM, 9 αPIF Ab and PIF 10.5 nM, 10 αPIF Ab {circle around (R )} andPIF 16.8 nM); Statistical analysis: Difference in means between groupswas determined by one-way ANOVA, followed by Tukey's post-test.Differences from control are indicated as b, p<0.01; c, p<0.001.Differences between anti-receptor 10 mg/ml and PIF are indicated as d,p<0.05; e, p<0.01 and f, p<0.001;

FIG. 10C shows a graphical representation of expression of p42 in C₂C₁₂myotubes in response to PIF and anti-receptor antibody (10 μg/ml) (Lanelegend: 1 PBS control, 2 PIF 2.1 nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM, 5 PIF16.8 nM, 6 αPIF Ab control, 7 αPIF Ab and PIF 2.1 nM, 8 αPIF AB and PIF4.2 nM, 9 αPIF Ab and PIF 10.5 nM, 10 αPIF Ab {circle around (R )} andPIF 16.8 nM); Statistical analysis: Difference in means between groupswas determined by one-way ANOVA, followed by Tukey's post-test.Differences from control are indicated as c, p<0.001. Differencesbetween anti-receptor 10 mg/ml and PIF are indicated as f, p<0.001;

FIG. 10D shows a graphical representation of expression of E2_(14k) inC₂C₁₂ myotubes in response to PIF and anti-receptor antibody (10 μg/ml)(Lane legend: 1 PBS control, 2 PIF 2.1 nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM,5 PIF 16.8 nM, 6 αPIF Ab control, 7 αPIF Ab and PIF 2.1 nM, 8 αPIF ABand PIF 4.2 nM, 9 αPIF Ab and PIF 10.5 nM, 10 αPIF Ab {circle around (R)} and PIF 16.8 nM); Statistical analysis: Difference in means betweengroups was determined by one-way ANOVA, followed by Tukey's post-test.Differences from control are indicated as a, p<0.05; b, p<0.01.Differences between anti-receptor 10 mg/ml and PIF are indicated as d,p<0.05 and e, p<0.01;

FIG. 10E represents an Actin blot as a loading control to show thatequal amounts of protein (PIF and anti-receptor antibody, 10 μg/ml) havebeen loaded in FIG. 10A to FIG. 10D. (Lane legend: 1 PBS control, 2 PIF2.1 nM, 3 PIF 4.2 nM, 4 PIF 10.5 nM, 5 PIF 16.8 nM, 6 αPIF Ab control, 7αPIF Ab and PIF 2.1 nM, 8 αPIF AB and PIF 4.2 nM, 9 αPIF Ab and PIF 10.5nM, 10 αPIF Ab and PIF 16.8 nM);

FIG. 11A Weight change in MAC16 tumour bearing mice treated daily i.p.with and without Anti-PIF IgG; Statistically significant c, P<0.001 fromcontrol by one-way ANOVA followed by Tukey's post-test; B Tumour Volumein MAC16 tumour bearing mice treated with and without Anti-PIF IgG;

FIG. 12A Protein synthesis in the soleus muscle of MAC16 Tumour bearingmice treated with and without Anti-PIF IgG; Statistically significant a,P<0.05 from control by one-way ANOVA followed by Tukey's post-test;

FIG. 12B shows soleus muscle weight in proportion to body weightcompared with solvent treated controls; Statistical analysis: Differencein means between groups was determined by one-way ANOVA, followed byTukey's post-test. Differences from solvent are indicated as b, p<0.01;

FIG. 12C is a graphical representation of Tyrosine release assay resultsfor PIF receptor antibody (3.47 mg/kg) in vivo; Statistical analysis:Difference in means between groups was determined by one-way ANOVA,followed by Tukey's post-test. Differences from solvent are indicated asc, p<0.001;

FIG. 12D shows the effect of anti-receptor antibody (3.47 mg/kg) onchymotrypsin-like enzyme activity in gastrocnemius muscle; Statisticalanalysis: Difference in means between groups was determined by one-wayANOVA, followed by Tukey's post-test. Differences from solvent areindicated as a, p<0.05 and b, p<0.01;

FIG. 13A shows a graphical representation of expression of 20Sproteasome α-subunits in gastrocnemius muscle in response toanti-receptor antibody (3.47 mg/kg); Statistical analysis: Difference inmeans between groups was determined by one-way ANOVA, followed byTukey's post-test. Difference from control is indicated as c, p<0.001.Difference between anti-receptor and MAC16 group is indicated as f,p<0.001;

FIG. 13B shows a graphical representation of expression of MSS1 ingastrocnemius muscle in response to anti-receptor antibody (3.47 mg/kg);Statistical analysis: Difference in means between groups was determinedby one-way ANOVA, followed by Tukey's post-test. Difference from controlis indicated as b, p<0.01. Difference between anti-receptor and MAC16group is indicated as d, p<0.05;

FIG. 13C shows a graphical representation of expression of p42 ingastrocnemius muscle in response to anti-receptor antibody (3.47 mg/kg);Statistical analysis: Difference in means between groups was determinedby one-way ANOVA, followed by Tukey's post-test. Difference from controlis indicated as a, p<0.05. Difference between anti-receptor and MAC16group is indicated as e, p<0.001;

FIG. 13D shows a graphical representation of expression of E2_(14k) inresponse to anti-receptor antibody (3.47 mg/kg); Statistical analysis:Difference in means between groups was determined by one-way ANOVA,followed by Tukey's post-test. Difference from control is indicated asb, p<0.01. Difference between anti-receptor and MAC16 group is indicatedas e, p<0.001;

FIG. 13E shows a representation of Myosin expression in gastrocnemiusmuscle in response to anti-receptor antibody (3.47 mg/kg); Statisticalanalysis: Difference in means between groups was determined by one-wayANOVA, followed by Tukey's post-test. Difference from control isindicated as b, p<0.01. Difference between anti-receptor and MAC16 groupis indicated as d, p<0.05;

FIG. 13F represents an actin control loading blot expression ingastrocnemius muscle in response to anti receptor (3.47 mg/kg) to showthat equal amounts of protein have been loaded in FIG. 13A to FIG. 13E.

EXAMPLE 1

The invention is based upon the following experiments that wereconducted to investigate, and characterise PIF binding sites in varioustissues.

Materials and Methods

Chemicals: Bovine fetal serum, RPMI1640 and Dulbecco's Modified EaglesMedium (DMEM) were purchased from GIBCO-BRL (Scotland, United Kingdom).

MAC16 monoclonal antibody was isolated from the culture medium of ahybridoma cell line (Todorov, P. T., McDevitt, T. M., Cariuk, P., Coles,B., Deacon, M. and Tisdale, M. J. Induction of muscle proteindegradation and weight loss by a tumor product. Cancer Res., 56:1256-1261, 1996.) using a protein A-Sepharose column.

L-[2,6-³H] phenylalanine (specific activity 54 Cimmol⁻¹) and Na₂ ³⁵SO₄(specific activity 10-100 mCimmol⁻¹) were purchased from Amersham Int.,(Buckinghamshire, United Kingdom).

All chemicals were purchased from Sigma Chemical Co., (Dorset, UnitedKingdom). Optiphase Hisafe 3 scintillation fluid was supplied by Fisons(Loughborough, United Kingdom).

Cell Culture and Tumor Propagation: The C₂C₁₂ mouse myoblast cell linewas grown in 60×15 mm petri dishes in 3 ml DMEM supplemented with 12%fetal bovine serum, 1% non-essential amino acids and 1%penicillin-streptomycin in a humidified atmosphere of 5% CO₂ in air at37° C. All experiments with myoblasts were performed on cells in thesubconfluent state. MAC16 cells were maintained in RPMI 1640 mediumcontaining 5% fetal bovine serum at 37° C. under an atmosphere of 5% CO₂in air. Normal human muscle cells, Hs94MU, were obtained from theEuropean Collection of Cell Cultures (Wiltshire, United Kingdom) andwere maintained in Dulbecco's Modified Eagles medium containing 2 mMglutamine and 10% fetal bovine serum under an atmosphere of 5% CO₂ inair. For biosynthetic labeling the cell suspension contained Na₂ ³⁵SO₄(1 μCiml⁻¹) for 48 h in RPMI 1640 medium containing 1.5% dialyzed fetalbovine serum.

Pure strain NMRI mice, bred in the inventors' own colony, were implantedin the flank with fragments of the MAC16 tumor, excised from donoranimals with established weight loss. Weight loss is evident 10-12 daysafter transplantation, when the tumor becomes palpable. Fragments of theMAC13 tumor were implanted by the same procedure. This tumor produces noweight loss during growth.

Purification of Labeled PIF Cells: were sedimented by low speedcentrifugation (1500 rpm for 5 min on a bench-top centrifuge). The cellpellet was resuspended in 1 ml of 10 mM Tris. HCl, pH 8.0, containing0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM EGTA and 1 mMdithiothreitol and dissociated using an ultrasonic oscillator. Aftercentrifugation (15,000 rpm for 20 min) solid ammonium sulfate (38% w/v)was added slowly to the supernatant with stirring, and the mixture wasstored overnight at 4° C. Salt was removed from the sample byultrafiltration with an Amicon filtration cell containing a membranefilter with a molecular weight cut-off of 10,000 against the sonicatingbuffer. The concentrated sample was loaded onto an affinity columncontaining MAC16 monoclonal antibody (Todorov, et al. Cancer Res. supra)coupled to Affi-Gel Hz (Bio-Rad, Hemel Hempstead, United Kingdom)equilibrated with 10 mM Tris. HCl, pH 8.0. After overnight circulationat a flow rate of 5 ml h⁻¹, the column was washed with 10 mM Tris. HCl,pH 8.0, and the retained material was eluted with 100 mM glycine HCl, pH2.5. After neutralization with 1M Tris. HCl, pH 8.0, the fractionscontaining radioactivity were concentrated by Amicon filtration againstwater and further purified by hydrophobic chromatography using aBrownlee Aquopore RP-300 C₈ column and an acetonitrile in water gradientas described (Todorov, et al. Cancer Res. supra and Todorov, et al.Nature. supra). Material eluting at 55% acetonitrile was concentratedagainst water using an Amicon filtration cell containing a membranefilter with a molecular weight cut-off of 10,000.

Membrane Isolations: Sarcolemma membranes were prepared fromgastrocnemius muscle of mice bearing either the MAC16 or MAC13 tumoressentially as described (Ohlendieck, K., Ervasti, J. M., Snook, J. B.and Campbell, K. P. Dystrophin-glycoprotein complex is highly enrichedin isolated skeletal muscle sarcolemma. J. Cell Biol., 112: 135-148,1991). Briefly gastrocnemius muscle (5 g) was excised and homogenized in20 mM sodium pyrophosphate, 20 mM sodium phosphate, 1 mM MgCl₂, 0.303Msucrose, 0.5 mM EDTA, pH 7.0, containing the protease inhibitorsaprotinin (76.8 nM), leupeptin (1.1 μm), pepstatin A (0.7 μM), benzamide(0.83 mM), iodoacetamide (1 mM) and PMSF (0.23 mM). The homogenate wascentrifuged at 30,000×g for 30 min. Light mucrosomes were obtained fromthe supernatant after adding solid KCl to a final concentration of 0.6M,followed by centrifugation at 142,000 g for 35 min. The pellets weresuspended in 0.303M sucrose, 20 mM Tris-maleate, pH 7.0 (buffer B),treated again with KCl, followed by centrifugation as described. Thefinal pellets of light microsomes were resuspended in buffer B (6 ml)containing 0.6M KCl and 1 ml aliquots were loaded onto 7 ml of 0.878Msucrose, 0.6M KCl, 20 Mm Tris-maleate, pH 7.0 in centrifuge tubes andcentrifuged at 112,000 g for 17 h. The crude surface membrane fractionat the 0303M/0.878M sucrose interface was collected and resuspended inbuffer B and stored frozen at −80° C. Sarcolemma membranes from pigmuscle were prepared by the same procedure.

For C₂C₁₂ Cell Membranes: homogenization was carried out in 20 mM HEPES,pH 7.4, 1 mM EDTA, 0.5 mM PMSF and 1 mM DTT at 4° C. The homogenate wascentrifuged at 20,000 rpm for 30 min and washed with the same buffer.The pellet was used for binding studies. Adipocyte plasma membranes wereprepared from adipocytes isolated from epididymal adipose tissue of maleBKW mice by a modification of the protocol of Belsham et al. (Belsham,G. J., Denton, R. M. and Tanner, M. J. A. Use of a novel rapidpreparation of fat-cell plasma membranes employing percoll toinvestigate the effects of insulin and adrenaline on membrane proteinphosphorylation within intact fat cells. Biochem. J., 192: 457-467,1980.). Essentially plasma membranes were isolated from other componentsof a cell homogenate using a self-forming Percoll gradient. The membranefractions were washed in a NaCl buffer, diluted in 10 mM Tris. HCl, pH7.4, 250 mM sucrose, 2 mM EGTA and 4 μM PMSF at 1-2 mg ml⁻¹, snap frozenin liquid nitrogen and stored at −70° C. until use. Hepatocyte plasmamembranes were purified by a scheme similar to that for adipocytes(Belsham et al. Supra), which had been modified for hepatocytes(Nakamura, T., Tomomura, A., Noda, C., Shimoji, M. and Ichihara, A.Acquisition of a β-adrenergic response by adult rat hepatocytes duringprimary culture. J. Biol. Chem., 258: 9283-9289, 1983).

Binding Studies: Membranes (200 μg protein suspended in 200 μl PBS) wereincubated for 24 h at 4° C. with various concentrations of [³⁵S]PIF in50 μl PBS as detailed in the figure legends. Bound and freeradioactivity was separated by centrifugation for 5 min at 13,000 g.

Determination of Affinity Constant (Kaff) of PIF for MonoclonalAntibody: The affinity of an antibody for its antigen can be estimatedif bound and free antigen can be measured when a fixed trace amount ofantigen is allowed to bind to serial dilutions of antibody. The antibodyconcentration at half-maximal binding is a measure of affinity.

Monoclonal antibody was purified from the tissue culture supernatant ofa hybridoma as described (Todorov, et al. Cancer Res. supra) using aProtein A column. Serial dilutions of the antibody were made in thedilutent 0.25M Tris-HCl, pH 8.5 with 2% calf serum and 1% Tween 20 to afinal dilution factor of 10⁴-10⁵. PIF was iodinated as described((Todorov, et al. Nature. supra) and diluted in the above diluent suchthat 50 μl contained 2×10⁴ cpm. The diluted monoclonal antibody (100 μl)was dispensed into tubes followed by ¹²⁵I PIF (50 μl) and the tubes wereincubated for 2 h at room temperature. Then Protein A-sepharose (100 μl)was added and the tubes were shaken for a further 2 h. Dilutent (3 ml)was added and the tubes were centrifuged, decanted and washed withanother 3 ml of dilutent. The final sedimented solid phase was countedin a gamma counter.

Competitive Binding of [³⁵S]PIF. C₂C₁₂ membranes (200 μg) in PBS (250μl) were incubated with 1, 5, 10, 50, 100, 500 or 1000 ng of eithermonoclonal antibody, chondroitin, dermatan or heparan sulfate and 20 nM(480 ng) of [³⁵S]PIF overnight at 4° C. The bound radioactivity wasdetermined from the radioactivity in the pellet obtained bycentrifugation at 13,000 g for 5 min.

The Kaff was determined according to a modification (Clark, B. R. andTodd, C. W. Avidin as a precipitant for biotin-labelled antibody in aradioimmunoassay for carcinoembryonic antigen. Anal. Biochem., 121:257-262, 1982.) of the method of Muller (Muller, R. Calculation ofaverage antibody affinity in anti-hapten sera from data obtained bycompetitive radioimmunoassay. J. Immunol. Methods, 34: 345-352, 1980.).

Kaff=^(I)/(I _(t) −T _(t)) (1−1.56+0.5b ²)

where I_(t) is the inhibitor concentration at 50% inhibition of PIFbinding; T_(t) the total PIF concentration; b the fraction of PIF boundin the absence of inhibitor.

Measurement of Protein Breakdown. C₂C₁₂ myoblasts were labeled withL-[2,6-³H]phenylalanine (0.5 μCi specific activity 0.72 Ci mmol⁻¹) for24 h. After labeling, cells were washed and incubated in fresh medium (3ml) in the presence of PIF and cycloheximide (1 μM) for the requiredtime, and the amount of radioactivity released into the medium wasmeasured. Protein bound radioactivity was determined by washing thecells three-times with ice-cold PBS (1 ml, pH 7.4) and after removal ofany residual PBS incubation was continued at 4° C. for 20 min with 0.2Mperchloric acid (1 ml). The perchloric acid was removed and replacedwith 1 ml of 0.3M NaOH at 4° C. for 30 min, followed by a furtherincubation at 37° C. for 20 min. The NaOH solution containing thedissolved cellular proteins was transferred to clean tubes and a further1 ml of 0.3M NaOH was used to rinse the dishes. The rate of proteolysiswas calculated by dividing the radioactivity released into theincubation medium by the protein-bound radioactivity.

Results

Binding studies have been conducted using [³⁵S]PIF, obtained bybiosynthetically labeling MAC16 cells. The radioligand was purified fromcell supernatants using a combination of affinity chromatography,followed by reverse phase hplc on a C₈ column. Ligand binding studieswere performed using membranes isolated from the murine myoblast cellline C₂C₁₂ (results not shown) and the human muscle cell line Hs 94 MU(results not shown). In both species Scatchard analysis of the bindingreaction provided evidence for two binding sites with Kd˜10⁻¹⁰M and10⁻⁹M (Table 1). Similar binding sites were observed in sarcolemmamembranes isolated from the pig (results not shown). In sarcolemmamembranes isolated from the gastrocnemius muscle of NMRI mice bearingthe MAC16 (results not shown) and MAC13 tumors (results not shown), aswell as liver plasma membranes (Table 1), two binding sites were alsoobserved, with the K_(d) of the lower affinity binding site beingreduced from 10⁻⁹ to 10⁻¹⁰M. There was no evidence for upregulation ofreceptor number in mice bearing the MAC16 tumor with cachexia incomparison with that found in sarcolemma membranes from mice bearing theMAC13 tumor, which does not induce cachexia. Plasma membranes fromsoleus muscle and heart also showed evidence for two binding sites forPIF (Table 1), while no receptor was detected on kidney or adiposetissue.

TABLE 1 Affinity Constants for Binding of PIF to Plasma Membranes TissueSource Kaff × 10⁻¹⁰ M Kaff₂ × 10⁻⁹ M C₂C₁₂ 1.4 1.2 Hs94MU 1.1 1.7 Pig4.7 8.2 Mouse gastrocnemius 5.2 0.1 muscle Mouse liver 6.9 0.2 Mousesoleus muscle 0.1  0.12 Mouse heart 3.0 2.3 Mouse adipose - nd - nd nd =non detectable

The biological activity of PIF is destroyed when the N- and O-linkedoligosaccharide chains are removed by incubation with peptide:N-glycosidase F (PNGase F) or endo-α-N-acetylgalactosaminidase(O-glycosidase). To determine the effect of deglycosylation of PIF onbinding to the receptor, experiments were conducted with peptide labeledPIF generated by incubating MAC16 cells with L-[2,5-³H] histidine. After24 h incubation with PNGase F or O-glycosidase binding of [³H] PIF wassubstantially reduced (FIG. 1), with only non-specific binding of thelabeled polypeptide chain to the membrane.

The affinity of binding of PIF to monoclonal antibody (Kaff 10⁸M⁻¹) wasfound to be less than binding to either high or low affinity sites onthe muscle receptor (results not shown). However, when the monoclonalantibody was added to C₂C₁₂ membranes at concentrations between 1 and1000 ng/250 μl binding to the receptor was effectively inhibited (Kd1.4×10⁻⁸M). The monoclonal antibody was less effective at competing withmembrane receptors for PIF, when added after PIF (Kd 5.8×10⁻⁷M).

Although PIF is a sulfated glycoprotein the oligosaccharide chains havesome similarity to a proteoglycan, since chondroitinase ABC destroys theantigenic determinants, and reduces the Mr, although no low molecularweight material corresponding to olisaccharides was obtained. Thissuggests that binding of PIF to the receptor may be attenuated byproteoglycans. To investigate this the effect of chondroitin, dermatanand heparan sulfate at concentrations between 5 and 5000 ng per assay onthe binding of PIF to receptors on C₂C₁₂ membranes has been determined.Of the three proteoglycans only chondroitin sulfate showed competitiveinhibition of binding (results not shown) with Kd 1.1×10⁻⁷M.

Ligand blotting of [³⁵S]PIF to triton solubilized membranes from C₂C₁₂cells electrophoresed in 15% SDS-PAGE and transferredelectrophoretically to nitrocellulose filters provided evidence for abinding protein with apparent Mr 40,000 (FIG. 2A).

Increasing concentrations of non-labeled PIF were capable of displacingradioactivity from the binding proteins (FIG. 2B) confirming thatbinding to the receptor was specific.

Since C₂C₁₂ myoblasts possess receptors for PIF it was important toestablish functional activity in this cell line. The effect of PIF onprotein degradation was measured by the release ofL-[2,6-³H]phenylalanine in the presence of cycloheximide from cellspre-labeled for a 24 h period. An increased rate of protein degradationwas observed within 6 h after the addition of PIF, which was maximal atconcentrations between 0.98 and 1.4 nM (results not shown), which isclose to the binding affinity of PIF to this cell line. Increasedconcentrations of PIF resulted in a decrease of phenylalanine releasesuggesting a negatively cooperative interaction between the two bindingsites. Increased rates of protein degradation were observed over longerperiods of time (24 and 48 h) at concentrations of PIF between 0.14 and1.4 nM.

Discussion

In order for PIF to induce protein degradation in skeletal muscle theremust be a specific interaction with a muscle protein receptor capable oftranslating the message into activation of the intracellular proteindegradative system. Since PIF is a highly glycosylated and sulfatedglycoprotein, it is likely that this will be membrane bound. The resultsof the present study provide evidence for specific, high affinitybinding sites for PIF in muscle cells. The affinity of binding wascomparable with that found for insulin and showed 10-100-fold greateraffinity than binding to a monoclonal antibody, which has been utilisedin the purification of PIF. However, high concentrations of the antibodywere capable of displacing PIF from the membrane receptor. This wouldexplain why high concentrations of the antibody were required toneutralise the biological effect of PIF. As with binding of PIF to theantibody, binding to the receptor is probably mediated through thesulfated oligosaccharide chains, since binding was specificallyinhibited by chondroitin sulfate, but not by the related proteoglycansdermatan and heparan sulfate. In addition enzymatic deglycosylationresulted in the loss of specific binding of PIF to the receptor. Thehigh affinity binding probably results from electrostatic interactionbetween PIF and the receptor.

A Scatchard plot of binding of PIF to muscle membranes from mouse, pigand man was nonlinear, indicating either two discrete sites, orcooperative interactions between the binding sites. Ligand blotting of[³⁵S]PIF to triton solubilized membranes from C₂C₁₂ cellselectrophoresed in 15% SDS-PAGE, provided evidence for a binding proteinof apparent Mr 40,000. Curvilinear Scatchard plots of steady statebinding have been described for a number of hormonal and nonhormonalsystems. For insulin this has been shown to represent negativecooperativity in the binding sites. This provides a mechanism in whichbinding to the receptor is favoured at low concentrations of thehormone, but becomes more difficult as the concentration of the hormoneis increased. Such an effect is apparent for protein degradation inC₂C₁₂ cells induced by PIF, where a bell-shaped dose-response curve wasobserved. Previous studies have suggested similar dose-response curvesfor protein degradation in isolated soleus and gastrocnemius musclesinduced by PIF. In addition the increased protein breakdown intumor-bearing animals has been reported to decrease as the tumor sizeincreases. These results suggest negative cooperative interactionsbetween the PIF binding sites.

We have recently confirmed that PIF is responsible for the loss ofskeletal muscle in mice bearing the MAC16 tumor with cachexia. However,the number of binding sites for PIF in skeletal muscle was comparable inmice bearing the MAC16 tumor and the MAC13 tumor, which does not inducecachexia, suggesting that the induction of muscle protein degradationduring the process of cachexia is not due to upregulation of receptors.Instead it appears to be related to the production of PIF by the tumor,since urinary analysis showed PIF to be present only in cancer patientswith weight loss, and not in those who were weight stable, or innon-cancer patients. Thus production of PIF by the tumor leads toconstitutive activation of muscle protein degradation.

The distribution of the PIF receptor in tissues is commensurate with arole for PIF in mediating catabolism of skeletal muscle proteins.Protein degradation rates in C₂C₁₂ myoblasts were shown to increase by50-90% in response to PIF, with maximal stimulation at a concentrationof 0.98-1.4 nM, which is close to the binding affinity of PIF to thereceptor. The role of PIF receptors in the liver is not known, since theliver responds to PIF by increasing in weight, rather than undergoingproteolysis as observed in skeletal muscle. This suggests that the PIFreceptor in liver is not coupled to the second messenger system, whichresults in activation of proteolysis. The receptor may be used forremoving PIF from the circulation or may result in activation of othersystems, e.g., the acute phase response in hepatocytes.

Our studies to date have only identified PIF in association with cancercachexia and not with other weight losing conditions. It is thusinteresting to find muscle possessing a receptor for such a factor, evenwithout prior exposure to PIF. The binding affinity and molecular weightof the receptor appear to be similar in mouse, pig and man suggesting auniversality of function across the species. The natural agonistregulating skeletal muscle catabolism, e.g., during dietary deficiencyor TNF-α-induced catabolic changes, is unknown, but may resemble PIF tothe extent of cross-reactivity of receptors. Little is known about theintracellular processes controlling protein catabolism in skeletalmuscle, but the end-result appears to be activation of theATP-ubiquitin-dependent proteolytic system.

EXAMPLE 2

Having characterised PIF Binding sites (see Example 1), the inventorsproceeded to isolate and sequence the PIF receptor according to thefirst aspect of the invention.

Isolation of the PIF Receptor from C₂C₁₂ Myotubes

The inventors have established that Wheat Germ Agglutinin (WGA) (whenlinked to sephadex) will bind the oligosaccharide chains of PIF. Thisenabled them to effectively isolate the PIF receptor, after incubationwith PIF, followed by lectin chromatography on WGA.

C₂C₁₂ membrane samples were prepared by sonicating in receptor buffer(20 mM HEPES pH 7.4, 1 mM EDTA, 0.5 mM PMSF, 1 mM DTT at 4° C.) andcentrifuging at 20,000 rpm for 20 min. The pellet was washed andsolubilized in 1% Triton for 30 min. The sample was then dialysedagainst PBS overnight at 4° C. 200 μl solubilized, dialysed sample wasincubated with ³⁵S PIF for 24 h at 4° C. in the presence of proteaseinhibitors, after which the PIF receptor was purified using a WGAcolumn. The column (1 ml bed, 10 mg WGA/ml) was loaded with sample andwashed with 20 volumes of wash buffer (10 mM Tris pH 7.4 with 0.02%NaN₃). Elution of the receptor was by 0.1M N-acetylglucosamine in washbuffer. 10 fractions (1 ml) were collected and stored at 4° C. inpresence of protease inhibitors.

The radioactive fractions were concentrated using a Microcon centrifugewith a membrane to cut off proteins with a molecular mass less than 10kDa, and were electrophoresed on 15% SDS-PAGE (FIG. 3). The receptorappeared as a single protein of apparent Mr 40 kDa. A similar molecularmass was evident from exclusion chromatography using Sephadex G-50(results not shown). The PIF-receptor complex ran as a single fraction.A similar result was obtained after cross-linking of PIF to the receptorusing glutaraldehyde (results not shown). Control incubations in whichsolubilized membranes isolated from C₂C₁₂ myotubes were subjected tolectin chromatography on WGA without prior incubation with PIF showedthat no protein was eluted, confirming that the 40 kDa material was notan endogenous glycoprotein.

Sequence Analysis of PIF Receptor (Edman)

N-Terminus

(SEQ ID No. 1) DINGGGATLPQPLYQTAAVLTAGFA

This sequence matches a peptide fragment from a synovial fluid proteinp205, with T-cell stimulatory activity (J. Immnuol.; (1996) 157;1773-80).

(SEQ ID No. 13) DINGGGATLPQKLYLIPNVL

This further sequence is believed to represent a polymorphic variantreceptor.

Internal Peptide Fragments

(SEQ ID No. 2) TAINDTFLNADSNLSIGK (SEQ ID No. 3) XATVAGVSPAPANVSAAIGA(SEQ ID No. 4) . . . IIPATTAGE . . . (SEQ ID No. 5). . . TYMSPDYAAATLAG . . . (SEQ ID No. 6) FVPLPT (SEQ ID No. 7)TELSNYVTAXGTxxG (SEQ ID No. 8) VTTAGSDS (SEQ ID No. 9) DVNGG(SEQ ID No. 10) LTTWDLIADSGR

There is no sequence homology of the internal peptides with otherproteins in the database.

EXAMPLE 3

Having sequenced the PIF receptor the inventors proceeded to developagents for use according to the fourth, fifth or sixth aspects of theinvention.

The inventors established that the peptide of SEQ ID No.1 was able toblock PIF binding to the PIF receptor and thereby demonstrated that thepeptide may be used as an agent according to the fourth, fifth or sixthaspects of the invention.

Methods

The techniques employed in the purification of PIF and proteindegradation assay, ‘chymotrypsin-like’ enzyme activity and Westernblotting are described above and also contained in the followingpublications:

-   -   1. Gomes-Marcondes et al., Br. J. Cancer (2002) 86, 1628-1633.    -   2. Whitehouse and Tisdale, Br. J. Cancer (2003) 89, 1116-1122.    -   3. Smith and Tisdale, Br. J. Cancer (2003) 89, 1783-1788.

Results

PIF induced protein degradation in murine myotubes with a bell-shapeddose-response curve as previously reported (Gomes-Marcondes et al.supra) (FIG. 4). This effect was completely attenuated by the N-terminalsynthetic peptide at a concentration of 10 μM. At this concentration thepeptide also blocked the PIF-induced increase in chymotrypsin-likeenzyme activity (FIG. 5), the predominant proteasome proteolyticactivity.

Western blotting showed that the peptide also completely prevented thePIF-induced increase in expression of 20S proteasome α-subunits (FIG.6A), two ATPase subunits of the 19S regulator of the proteasome MSS1(FIG. 6B), p42 (FIG. 6C) as well as the ubiquitin-conjugating enzyme,E2_(14k) (FIG. 6D). Inhibition of the induction of theubiquitin-proteasome proteolytic pathway by the peptide resulted inattenuation of the PIF-induced decrease in expression of themyofibrillar protein myosin (FIG. 6E). These results suggest that PIFbinds to the N-terminal region of the receptor peptide preventinginteraction with the receptor in the myotubes.

These data clearly show that the peptide of SEQ ID No 1 may be used asan agent according to the present invention.

EXAMPLE 4

An antibody agent for use according to the fourth, fifth or sixthaspects of the invention was also investigated.

Methods

Polyclonal antiserum was generated to a 19-mer derived from the first 19amino acids of the N-terminal peptide fragment (SEQ ID No 1). Theantiserum was produced in a rabbit under the terms of a confidentialcontract with Severn Biotech Ltd., Worcs, UK.

In more detail, polyclonal antiserum was produced by conjugating the19mer peptide (5 mg) to 5 mg PPD (as carrier protein) with sulpho-SMCCthrough a C-terminal cysteine and subsequently immunising two rabbits bysubcutaneous injection of the antigen (50-200 mg) at 0.25 ml at each offour sites in Freud's adjuvant. Serum was supplied from a test bleed,production bleed and terminal bleed.

The antiserum detected the PIF receptor by Western blotting (FIG. 7)after purification of the antibody by adding 50% saturated ammoniumsulphate followed by Protein-A column chromatography (PURE1A kit, SigmaAldridge, Dorset, UK).

Methods employed in Examples 1-3 were otherwise employed.

Results

The effect of the antibody to the PIF receptor at concentrations between5 and 15 μg/ml on protein degradation induced by PIF is shown in FIG. 8.Partial attenuation of the PIF effect was seen at a concentration of 5μg/ml, while complete attenuation was seen at concentrations of 10 μg/mland higher. A similar effect was seen on the PIF-induced increase inchymotrypsin-like enzyme activity (FIG. 9).

Western blotting showed that in C₂C₁₂ myotubes, the anti-receptorantibody also completely prevented the PIF-induced increase inexpression of 20S proteasome α-subunits (FIG. 10A), two ATPase subunitsof the 19S regulator of the proteasome MSS1 (FIG. 10B), p42 (FIG. 10C)as well as the ubiquitin-conjugating enzyme, E2_(14k) (FIG. 10D). Thesedata clearly show that an antibody raised against the peptide of SEQ IDNo 1 may be used as an agent according to the present invention.

To evaluate the ability of the anti-receptor antibody to prevent muscleprotein degradation by PIF in vivo, mice bearing the cachexia-inducingMAC16 colon carcinoma, where PIF has been shown to be responsible forthe loss of skeletal muscle (Lorite et al, Br. J. Cancer (1998) 78,850-856), were treated daily by the i.p. administration of anti-PIFreceptor polyclonal antisera (3.47 mg/kg). After 3 days treatment micereceiving the anti-receptor antibody had a significantly reduced weightloss compared with solvent controls (FIG. 11A). The effect on tumourvolume is shown in FIG. 11B). There was a significant effect on proteinsynthesis (FIG. 12A) and a significant increase in the weight of thesoleus muscle compared with solvent treated controls and this was notsignificantly different from weight matched NMRI mice without tumour(FIG. 12B). The anti-receptor antibody treatment attenuated proteindegradation in soleus muscle down to that of non tumour-bearing mice(FIG. 12C), as well as functional proteasome activity, as measured bythe chymotrypsin-like enzyme activity (FIG. 12D). Expression of 20Sproteasome α-subunits (FIG. 13A), MSS1 (FIG. 13B), p42 (FIG. 13C) andE2_(14k) (FIG. 13D) were all attenuated down to levels found in nontumour-bearing mice after treatment of cachectic mice with theanti-receptor antibody. The results in FIG. 13E show that theanti-receptor antibody reverses the loss of myosin in gastrocnemiusmuscle a seen in mice bearing the MAC16 tumour, up to levels found innon tumour-bearing mice. These data clearly show that an antibody raisedagainst the peptide of SEQ ID No 1 may be used as an agent according tothe present invention.

1-26. (canceled)
 27. A method of screening a compound to test whether ornot the compound has efficacy for treating cachexia, comprising:exposing cells or membranes comprising a receptor for ProteolysisInducing Factor (PIF), wherein the N terminus of the mature nativereceptor has the amino acid sequence of SEQ ID NO: 13 to a test compoundfor a predetermined length of time; (ii) detecting the activity orexpression of the receptor; and (iii) comparing the activity orexpression of the receptor in the cells or membranes treated with thecompound relative to activity or expression found in control cells ormembranes that were not treated with the compound wherein compounds withefficacy for treating cachexia decrease activity or decrease expressionof the receptor relative to the controls.
 28. A method of screening acompound, to test whether or not the compound causes cachexia,comprising: (i) exposing cells or membranes comprising a receptor forProteolysis Inducing Factor (PIF), wherein the N terminus of the maturenative receptor has the amino acid sequence of SEQ ID NO: 13 to a testcompound for a predetermined length of time; (ii) detecting the activityor expression of the receptor; and (iii) comparing the activity orexpression of the receptor in the cells or membranes treated with thecompound relative to activity or expression found in control cells ormembranes that were not treated with the compound; wherein compoundsthat cause cachexia increase activity or increase expression of thereceptor relative to the controls.
 29. An in vivo method as claimed inclaim 27 or claim 28 in which cells present in any experimental animalare exposed to the test compound. 30-35. (canceled)
 36. A method for thetreatment of cachexia, which method comprises administering to a subjectin need of such treatment a therapeutically effective amount of an agentthat specifically binds to the receptor for Proteolysis Inducing Factor(PIF) and decreases the biological activity of the receptor, wherein theN terminus of the mature native receptor has the amino acid sequence ofSEQ ID NO: 13, wherein the agent is a peptide or peptide derivative. 37.A method as claimed in claim 36 wherein the agent is a peptide fragmentof said receptor for PIF.
 38. A method as claimed in claim 36 whereinthe peptide fragment is an N terminal fragment of the receptor.
 39. Amethod as claimed in claim 36 wherein the agent is a peptide derivativehaving an increased half-life in vivo compared to the receptor fragmentfrom which it was derived.
 40. A method for the treatment of cachexia,which method comprises administering to a subject in need of suchtreatment a therapeutically effective amount of an agent that decreasesthe expression of the receptor for Proteolysis Inducing Factor (PIF),wherein the N terminus of the mature native receptor has the amino acidsequence of SEQ ID NO: 13, wherein the agent is selected from the listconsisting of: (i) an antisense DNA or RNA molecule that will bind toendogenous receptor RNA transcripts such as to reduce the receptorexpression; (ii) a short interfering RNA (siRNA) molecule or a shorthairpin RNA (shRNA) which results in the sequence-specific destructionof mRNA encoding the receptor.
 41. A method as claimed in claim 40wherein the agent is an antisense molecule selected from:(SEQ ID NO. 12) 5′GGCGAAGCCGGCGGTCAGCACGGCGGCGGTCTGGTACAGGGGCTGGGGCAGGGTGGCGCCGCCGCCGTTGATGTC . . . 3′ (SEQ ID NO. 15)5′CAGCACGTTGGGGATCAGGTACAGCTTCTGGGGCAGGGTGGCGCCGCCGCCGTTGATGTC . . . 3′.


42. A method as claimed in claim 40 wherein the agent is siRNA whichcomprise 10-50 nucleotides, 18-30 nucleotides, 21-25 nucleotides, or 21nucleotides.
 43. A method as claimed in claim 40 wherein the agent issiRNA which comprises double stranded RNA of 21 nucleotides length,optionally with a 2-nucleotide overhang at each 3′ end.
 44. A method asclaimed in claim 40 wherein the agent is shRNA having sense andantisense sequences connected by a hairpin loop.
 45. A method for thetreatment of cachexia, which method comprises administering to a subjectin need of such treatment a therapeutically effective amount of an agentthat specifically binds to the receptor for Proteolysis Inducing Factor(PIF) and blocks receptor-mediated intracellular signalling of thereceptor, wherein the N terminus of the mature native receptor has theamino acid sequence of SEQ ID NO: 13, wherein the agent is an antibodyor functional derivative of an antibody capable of binding specificallyto the receptor.
 46. An method as claimed in claim 45 wherein the agentis an antibody which has been raised against any one of peptides SEQ IDNo. 1-10 or
 13. 47. An method as claimed in claim 45 wherein the agentis a polyclonal antibody.
 48. An method as claimed in claim 45 whereinthe agent is a monoclonal antibody.
 49. An method as claimed in claim 45wherein the agent is a γ-immunoglobulin (IgG).
 50. A method as claimedin claim 45 wherein the agent is a functional derivatives of an antibodyhaving at least the variable domain thereof.
 51. A method as claimed inclaim 50 wherein the functional derivative is an antibody fragment. 52.A method as claimed in claim 51 wherein the antibody fragment is an scFVantibody.
 53. A method as claimed in claim 45 wherein the agent is ahumanised antibody.
 54. A method as claimed in claim 38 wherein the Nterminal fragment of the receptor is selected from or comprises SEQ IDNO. 1 or SEQ ID NO.
 13. 55. A method as claimed in claim 39 wherein thepeptide derivative is a peptoid or retropeptoid or a peptide whichcomprises D-amino acids.